Primer used in RT-PCR detection of chicken infectious bronchitis virus, kit comprising the primer and detection method thereof

A detection kit and technology for bronchitis, applied in the direction of microbiological-based methods, biochemical equipment and methods, microbiological measurement/inspection, etc., can solve the problem that the copy number of IBV genome cannot be truly reflected, and achieve large sample size and specificity Good performance, the effect of reducing the possibility of pollution

Inactive Publication Date: 2015-11-11
JIANGSU INST OF POULTRY SCI +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The fluorescent quantitative RT-PCR detection method of chicken infectious bronchitis virus reported in the above-mentioned literature uses detection primers all designed according to the gene sequence of IBV structural protein gene N. The copy number of virus N gene sequence in tissue and other samples is theoretically higher than the copy number of IBV genome. Therefore, when detecting fresh tissue samples, detection primers designed with structural genes such as N gene cannot truly reflect the copy number of IBV genome.

Method used

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  • Primer used in RT-PCR detection of chicken infectious bronchitis virus, kit comprising the primer and detection method thereof
  • Primer used in RT-PCR detection of chicken infectious bronchitis virus, kit comprising the primer and detection method thereof
  • Primer used in RT-PCR detection of chicken infectious bronchitis virus, kit comprising the primer and detection method thereof

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Embodiment 1

[0054] Design and synthesis of primers in the kit of the present invention in embodiment 1

[0055] According to the IBV genome sequence published in GenBank (GenBank accession number is FJ807652), select a highly conserved region with no special structure in the 5′ non-coding region sequence to design primers. The length of the primers is generally about 20 bases. There is no complementary sequence in:

[0056] Primer 1: 5’–CCGTTGCTTGGGCTACCTAGT-3’

[0057] Primer 2: 5'–CGCCTACCGCTAGATGAACC-3'; or

[0058] Primer 1: 5'-CTTTTGAGCCTAGCGTTGG-3'

[0059] Primer 2: 5’–TTGTCACTGTCTATTGTATGTCTGC-3’; or

[0060] Primer 1: 5’–GGCGGGTGTGTGGAAGTAGC-3’

[0061] Primer 2: 5'-GCCGACCTTGTGCGAGAACG-3'. Primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

Embodiment 2

[0062] Construction of embodiment 2 positive control plasmid

[0063] Preparation of biological materials: the chicken infectious bronchitis virus vaccine strain H120 used in the present invention was purchased from the China Veterinary Drug Administration; SPF chicken embryos were purchased from Beijing Meria Verton Company; JM109 genetically engineered bacteria and pMD18T vector were purchased from Dalian Bao Biology Engineering Co., Ltd.

[0064] RT-PCR amplification of the 5′ non-coding region sequence: primers were designed according to the genome sequence of infectious bronchitis virus H120 (GenBank accession number is FJ807652), used to amplify its 5′ non-coding region fragment, the primers were provided by Shanghai Synthesized by Sangon Bioengineering Technology Service Co., Ltd. The primer sequence is 5F: 5'-gttgctggtatcactgcttgt-3', 5R: 5'-GACCACTGGCATGCTGTT-3'. The H120 vaccine strain was inoculated into 11-day-old SPF chicken embryos, and the allantoic fluid wa...

Embodiment 3

[0066] The establishment of embodiment 3 SYBRGreenI real-time fluorescent quantitative RT-PCR

[0067] 1. Establishment of standard curve and analysis of melting curve

[0068] The positive control plasmid pMD5UTR was extracted and quantified using an endotoxin-free plasmid extraction kit (Omega, USA), and then diluted to 10 according to a 10-fold gradient. 0 ~10 9 copies / μL, positive plasmids with different dilutions were used as templates, and three replicates were set for each dilution, and real-time fluorescent quantitative PCR amplification of SYBRGreenI was performed, and a standard curve was drawn. The amplification reaction system is: SYBRGreenI, ROXReferenceDye, Ex Taq 10 μL of HSDNA polymerase, dNTPs, Mg2+, etc., 2 μL of pMD5UTR plasmid, 0.4 μL each of primer 1 and primer 2 at a concentration of 10 μmol / L, dH 2 O to make up to 20 μL. The amplification reaction program is: 95°C for 30s, then 95°C for 5s, 60°C for 20s for 40 cycles, then 95°C for 30s, 20°C / s, 6...

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Abstract

The invention relates to a primer used in RT-PCR detection of chicken infectious bronchitis virus, a kit comprising the primer and a detection method thereof. The primer includes a specific primer pair used for amplifying a 5-terminal non-coding region conserved sequence of chicken infectious bronchitis virus (IBV). The specific primer pair is composed of a primer 1 and a primer 2, wherein the primer 1 is a single-chain DNA represented in the Seq ID No.1, the Seq ID No.2 or the Seq ID No.3 in the sequence table, and the primer 2 is a single-chain DNA represented in the Seq ID No.4, the Seq ID No.5 or the Seq ID No.6 in the sequence table. Tests comprising specificity, sensitivity, stability and repeatability prove that the kit can only specifically amplify IBV nucleic acid and is excellent in linearity relationship within the range of 1*10<2>-1*10<7> copies/[mu]L. The sensitivity of the primer reaches 10<2> copies/[mu]L. The primer is suitable for quantitative detection of various tissue, cell, body fluid samples and vaccine production intermediate products.

Description

technical field [0001] The invention belongs to the field of veterinary microorganism detection, and relates to a chicken infectious bronchitis virus fluorescent quantitative RT-PCR detection kit, which is suitable for the quantitative detection of IBV nucleic acid in various tissues, cells, body fluid samples and vaccine production intermediate products. Background technique [0002] Infectious bronchitis (IB) is an acute and highly contagious disease caused by infectious bronchitis virus (IBV). The disease can invade tissues such as the trachea, kidney, oviduct and glandular stomach of chickens, causing the death of chicks, and the egg production and egg quality of laying hens are reduced. The prevalence of IB has caused huge economic losses to the poultry industry and seriously restricted the development of the poultry industry in the world. [0003] IBV belongs to the Gamma coronavirus of the Coronaviridae family, and its genome has a high mismatch rate due to its uniqu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 周生唐梦君许明陈连颐蒲俊华姜逸程旭沈欣悦李建梅戴亚斌戴有理赵宝华
Owner JIANGSU INST OF POULTRY SCI
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